Transcriptomic dissection of Intraepithelial Papillary Mucinous Neoplasms progression by spatial technologies identified novel markers of pancreatic carcinogenesis.

Abstract

Intraductal papillary mucinous neoplasms (IPMN) are one of the main precursor lesions of Pancreatic Ductal Adenocarcinoma (PDAC). The number of patients diagnosed with IPMN is constantly increasing. While in most of the cases IPMN present as indolent and nonmalignant entities, some degenerate into PDAC. The main mechanisms behind the IPMN progression to malignancy is still not fully understood. This is mainly due to the technological limit of the analyzes and to cysts heterogeneity whose malignant transformation potential is estimated based on size and degree of dysplasia without take in consideration the transformation time and therefore the real malignancy potential. Moreover, there is a general lack of consensus diagnostic markers to discern the Low-grade nonmalignant from High-grade malignant IPMN. In this study, we used two different Spatial Transcriptomic technologies (Visium, and GeoMx) to investigate the transcriptome of Low-grade dysplasia nonmalignant IPMN, Borderline IPMN, and High-grade dysplasia malignant IPMN to dissect the main mechanism that drives carcingenesis and to find specific markers associated to risk of tumor progression. We performed Visium spatial transcriptomics on two TMAs containing three Low-grade dysplasia nonmalignant IPMN, one Borderline IPMN, two High-grade dysplasia malignant IPMN, and two PDAC. We identified three specific epithelial cell clusters that characterize Low-grade dysplasia IPMN, Borderline IPMN, and High-grade dysplasia malignant IPMN and three transcription factors whose expression is associated with each grade. High-grade malignant IPMN were characterized by high expression levels of NKX6-2 and other markers of gastric isthmus cell lineage such as MUC5AC, PSCA, FERIL6. The SPDEF high IPMN cluster was found in Borderline IPMN and spotted in some regions of High-grade malignant IPMN. This cluster was characterized by high expression levels of SPDEF and other goblet cell lineage markers such as TFF2, AQP5, and MUC6. Low-grade nonmalignant IPMN were characterized by high expression levels of HOXB3, HOXB5, ZNF117. The association of these markers with the different grades was validated by GeoMx spatial transcriptomics on 43 additional IPMN samples divided according to their grade of dysplasia and malignancy. To better understand the transcriptomic changes along IPMN progression we performed spatial trajectory inference and we found that SPDEF high IPMN cluster cells are likely to evolve into NKX6-2 high malignant IPMN, and we found that this switch is characterized by the expression of NKX6-2 and other gastric markers. Taken together, the results presented here not only shed more light in to IPMN and PDAC oncogenesis, but also provided a plethora of novel malignancy-associated markers to be tested in diagnostic routine, to better delineate IPMN progression in patients and improve clinical management.